PROJECT SUMMARY Melioidosis caused Burkholderia pseudomallei is now recognized as a globally emerging pathogen infectious disease with high mortality (>40%), and it is severely underreported in 45 countries and never reported in another 34 countries with a large population at risk. Moreover, B. pseudomallei is listed as a Tier 1 bacterial biothreat/select agent by US CDC due to its potential use as a bioweapon. Current diagnostic methods/tools for melioidosis are limited and inefficient in rapidly, sensitively and reliably diagnosing melioidosis at the point of care (POC). Culture of B. pseudomallei from body fluids (taking 3 ~ 5 days) is still the gold standard for melioidosis diagnosis. Time-gated fluorescent (TGF) measurement using probes with long-lived fluorescence is a highly sensitive signal transduction method, and its coupling with simple and rapid POC lateral flow immunoassay (LFIA) is very attractive for melioidosis diagnosis. Since the same biomarker for melioidosis can be found in patient serum, whole blood, sputum, urine and pus, an ideal diagnostic tool is also expected to detect the biomarker in these complex biological media without sacrificing its analytical performance from one medium type to the other one. Such a feature will ensure the diagnostic tool's better applicability to various samples, and also can facilitate the R&D on the melioidosis diagnosis methodology (e.g., from invasive testing using blood/serum to noninvasive testing using urine/sputum/pus). However, highly sensitive TGF LIFA with a portable instrument for such purposes is not available. In this work, novel probes will be engineered to overcome the shortcomings of current time-gated probes. A portable, sensitive and cost-effective instrument for POC testing will be built for time-gated fluorescence measurement. Using the probes and the instrument, POC assays (i.e., TGF LFIAs) specific to melioidosis will be developed. Our probes conjugated with high affinity monoclonal antibodies as well as our instrument will offer POC testing on melioidosis with high sensitivity and specificity. Moreover, the quantitative data of our POC assays will facilitate more robust data analysis for accurate diagnosis, and also track the course of the disease for adjustment of the antibiotics doses or treatment times in melioidosis therapy. Our POC assays also have potential to be modified for other disease diagnosis.